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Molecular Biology Shared Resource
Dartmouth College
HB 7650
Remsen Building (Room 243)
Hanover, NH 03755-3526
Phone: 603-650-6546
Fax: 603-650-1129

DNA Sequencing Service General Information

Introduction

DNA sequencing is performed on the Applied Biosystems Model 3730, a capillary based electrophoresis system that uses fluorescent dye terminator chemistry. For details on the features of the instrument and chemistry, see the Applied BioSystems website.  We have you submit template and primer to the facility in the recommended amounts and we do the chemistry. The advantage is that your samples are treated uniformly and processed with control samples, using more efficient methods, and keeping costs down.

Sample Submission

Sequencing orders are placed through the CCOP's Core Server. Instructions for using the server are available here and in the download section of this website. Copies of your order with your sample(s) can be dropped off at any time at the laboratory in 241 Remsen, or the Borwell loading dock next to the Rubin elevator. Data from samples submitted before 10:30 AM at any site will be reported to you by the following morning except on weekends.

Data Distribution and Quality

When the sequencing is completed, you should receive an e-mail stating that your data is ready. Log back on to your CCOP's account and navigate to the Data Download tab. Using the Order ID number you will find an associated Zip file you can download which contains your data. Each sample will have two files associated with it. One file will show the electropherogram for your sample and the run and analysis conditions. You will also receive a file with a .seq extension. This file is a text file with raw sequence called by the computer. This file can be viewed using Microsoft Word. If this file is empty or contains only N's, your sample was unsuccessful.

We like the software FinchTV for viewing electropherograms it is available for free from the Geospiza website. FinchTV operates on both Mac and PC platforms and no conversion is needed. Free software is also avaliable from the Applied BioSystems web site Sequence Scanner 1.0 gives you the ability to aline a few sequences together along with other QA/QC features.

Always look at the data file and be sure your sample had a strong enough signal, proper base spacing and that the appropriate base caller was used. If you have sufficient non-problematic template, a good primer, and clean extension products, it is not unusual to get over 1000 bases of unambiguous data from our standard run. Once you develop some proficiency at editing the computer-called sequence, you can often get another 50+ bases. We cannot emphasize strongly enough that the quality of you sequence data depends on the purity and quantity of your template and the quality of your primer.

Last Updated: 3/5/14